The present invention refers to the use of biobalistic for introducing exogenous genes into a vegetable tissue and obtaining transgenic leguminous plants by regenerating the transformed tissue.
The use of genetic engineering techniques for introducing genes which are responsible for agronomic characteristics of interest may facilitate the development of new varieties of LEGUMINOSAE. The obtaintion of a transgenic plant requires methods of introducing the exogenous DNA into the vegetable tissue and regenerating the whole plant from such genetically transformed tissue. Depending upon the species to be transformed, various types of tissue have been used for the introduction of an exogenous DNA, the meristematic tissue been preferably employed in various transformation processes, primarily due to the ease regeneration of a plant from this type of tissue. Various processes have been proposed for introducing exogenous genes into apical meristematic cells of LEGUMINOSAE, among which the following can be pointed out: a) the Agrobacterium system; b) a system related to tissue electroporation and c) the biobalistic system. The introduction and integration of exogenous DNA into cells of LEGUMINOSAE have been demonstrated by various scientists and described in different publications such as (Aragxc3xa3o F. J. L., Grossi-de-Sxc3xa1 M. F., Almeida E. R, Gander E. S. Rech E. L. (1992); Particle bombardment mediated expression of a Brazil nut methionine-rich albumin in bean (Phaseolus vulgaris L.); Plant Molecular Biology 20:357-359. Lewis, M. F. and Bliss, F. A. (1994); Tumor formation and beta-glucuronidase expression in Phaseolus vulgaris L inoculated with Agrobacterium tumefactiens. Journal of the American Society for Horticultural Science; 119:361-366, Dillen W. Engler G. Van Montagu M. and Angenon G. (1995); Electroporation-mediated DNA delivery to seedling tissues of Phaseolus vulgaris L (common bean), Plant cell Reports, 15:119-124.
However, the low obtaintion frequency of the genetically transformed tissue, the low capacity of regenerating a fertile plant from said transformed tissue, together with the use of transformation methods the efficiency of which depends upon the genotype, have rendered it difficult to obtain transgenic leguminous plants (Brasileiro A. C. M.; Aragxc3xa3o F. J. L.; Rossi S. Dussi D. M. A.; Barros L. M. G.; and Rech E. L. (1996)xe2x80x94Susceptibility of common and tepary beans to Agrobacterium ssp. strains and improvement of Agrobacterium-mediated transformation using microprojectile bombardment J. Amer. Soc. Hort. Sci. 12:810-915 and Dillen W.; Van Montagu M. and Angenon G. (1995) Electroporation-mediated DNA delivery to seedling tissues of Phaseolus vulgaris L. (common bean). Plant Cell Reports 15:119-124).
With the development of the biobalistic process for the direct introduction of genes into vegetable cells at the end of the ""80 (Sanford J. C. Klein T. M., Wolf E. D. and Allen N. (1987) Delivery of substances into cell tissues using a particle bombardment process; Journal of Particle Science and Technology, 5:27-37), a great number of transgenic plants of several species have been obtained, including those species which proved to be recalcitrant to the transformation by the using other methods. This is due to the fact that it has become possible to introduce and express exogenous genes in any kind of vegetable tissue. Thus, any type of tissue having a potential ability to regenerate a whole fertile plant is suitable for transformation.
The biobalistic process was proposed by Sanford with a view to introduce genetic material into the nuclear genome of higher plants. Since then its universality of application has been appraised, and it has proved to be an effective and simple process for the introduction and expression of genes into bacteria, protozoa, fungi, algae, insects, vegetable and animal tissue, as well as isolated organcils as chloroplast and mitochondria, according to the results observed by Sanford J C, Smith F D and Russel J. A. (1993) Optimizing the biobalistic process for different biological application. Methods in Enzymology: 217:413-510. In the specialized literature there are several other examples of the use of biobalistic for the obtaintion of transgenic organisms such as, for instance, U.S. Pat. Nos. 5,565,346, 5,489,520 and WO 96/04392, among others.
In biobalistic microprojectiles accelerated at high speed are used for carrying and introducing nucleic acids and other substances into cells and tissues in vivo (Rech E. L. and Aragxc3xa3o F. J. L. (1997). The ballistics processxe2x80x94In: Brasileiro A. C. M. and Carneiro. V. T. C. (Ed)xe2x80x94Manual of genetic transformation of plants: EMBRAPA/Cenargen. This process has also been called as method of bombardment with microprojectiles, xe2x80x9cgene gunxe2x80x9d method, particle-acceleration method, among others. Different systems have been developed and constructed which are capable of accelerating microparticles (made of tungsten or gold), coated with nucleic acids sequences, at speeds higher than ink 1500 km/h-1. All these systems are based on the generation of a shock wave with enough energy for displacing a carrying membrane containing the microparticles coated with DNA. The shock wave can be generated by a chemical explosion (dry gunpowder), a discharge of helium gas under high pressure, by vaporization of a drop of water through a electric discharge at high voltage and low capacitance or at low voltage and high capacitance.
Those systems which use helium gas under high pressure and electric discharge have shown a wide spectrum of utilization. The accelerated particles penetrate the cellular wall and membrane in a non-lethal way, locating themselves randomly in the cellular organells. Then the DNA is dissociated from the microparticles by the action of the cellular liquid, and the process of integrating the exogenous DNA in the genome of the organism to be modified takes place (Yamashita T. lada, A. and Morikawa H. (1991)xe2x80x94Evidence that more than 90% of xcex2-glucuronidase-expressing cells after particle bombardment directly receive the foreign gene in their nucleus; Plant Physiol. 97:829-831).
In spite of the efficiency and universality of utilization of the biobalistic process, it depends upon the optimization of various physical and biological parameters, which is fundamental to the effective introduction of heterologous genes into a vegetable tissue.
For the obtaintion of transgenic plants from the apical region of embrionic axis, there are two essential requirements, namely: 1) introduction of exogenous genes with high frequencies into the cells of the apical regions, and integration of erogenous genes into the vegetable genome, and 2) regeneration and production of fertile transgenic plants from the resulting transformed cells.
With the development of the biobalistic process the in situ direct transformation of cells of the apical meristem is now possible. However, the development and further production of fertile transgenic plants require the regeneration and production of the plant from the transformed cells.
During the last few decades several attempts have been made to obtain the regeneration of fertile plants of commercially important LEGUMINOSAE. Although many advances have been achieved, no effectively positive results have been obtained yet. For instance, some methodologies of multiple shooting of apical and lateral meristems of embryos in different LEGUMINOSAE have been developed. However, these systems still present serious disadvantages.
Other regeneration systems developed for certain LEGUMINOSAE such as peanuts and soybeans involve the induction of somatic embryogenesis from mature and immature embryos cultivated at high doses of 2,4-D. However, the practical use of this system is limited since it is restricted to determined varieties, in addition to the fact that induction of unwanted genetic variations (somaclonal variation) also occurs with the consequent production of transgenic plants with their inherent agronomic characteristics changed.
Thus, the systems already known for the obtaintion of transgenic plants of LEGUMINOSAE based on the transformation of meristematic cells of the apical region, by using the biobalistic process present the disadvantages of impossibility of selecting the transformed cells, low production frequencies of transgenic plants and high frequency of chimeras (plants with an organ or groups of some transgenic cells and other non-transgenic cells).
It is therefore, the objective of the present invention to provide a process with high production frequency for the obtaintion of transgenic leguminous plants containing an exogenous DNA and which enables the selection of the transformed cells, the latter maintaining the agronomic characteristics of the plants from which they have originated.
The present invention refers to a process for producing transgenic leguminous plants containing exogenous DNA, which comprises the steps of:
a) introducing exogenous genes into cells of the apical meristem of the embryonic axis of leguminous plants by the biobalistic method:
b) inducing the multiple shooting of the cells in the apical meristematic region modified in step (a) by cultivating said embryonic axis in a medium containing a multiple shooting inducer; and
c) selecting the meristematic cells of the apical region as obtained in step (b) by further cultivation of said embryonic axis in a medium containing a molecule which concentrates in the apical meristematic region of said leguminous embryos.